Liver tissue engineering in the evaluation of drug safety

Abstract

Assessment of drug-liver interactions is an integral part of predicting the safety profile of new drugs. Existing model systems range from in vitro cell culture models to FDA-mandated animal tests. Data from these models often fail, however, to predict human liver toxicity, resulting in costly failures of clinical trials. In vitro screens based on cultured hepatocytes are now commonly used in early stages of development, but many toxic responses in vivo seem to be mediated by a complex interplay among several different cell types. We discuss some of the evolving trends in liver cell culture systems applied to drug safety assessment and describe an experimental model that captures complex liver physiology through incorporation of heterotypic cell-cell interactions, 3D architecture and perfused flow. We demonstrate how heterotypic interactions in this system can be manipulated to recreate an inflammatory environment and apply the model to test compounds that potentially exhibit idiosyncratic drug toxicity. Finally, we provide a perspective on how the range of existing and emerging in vitro liver culture approaches, from simple to complex, might serve needs across the range of stages in drug discovery and development, including applications in molecular therapeutics.

Figure 1. A live-dead stain of human and rat hepatocytes cultured in the scaffold alongside a schematic cross-section

(A). The scaffold resides within a well of the bioreactor (B) and there are 12 isolated 3D perfused bioreactors on each culture plate (C). Photograph courtesy of Karel Domansky, MIT 2008.

Figure 2. RT-PCR analysis of relative expression of Phase I and II drug metabolizing genes

Constitutive expression of Phase I and II genes in isolated rat hepatocytes, collagen sandwich culture (day 7) and 3D microreactor (day 7) cultures expressed as log twofold change relative to rat liver in vivo. Reproduced from Sivaraman et al. 2005 [117] with permission from Bentham Science Publishers Ltd (2009). RT-PCR: PCR after reverse transcription of RNA.

Figure 3. Measured clearance in 3D perfused culture of cryopreserved human hepatocytes compared with in vivo human CL for nine compounds: Sevidal, Kelly and Obach, manuscript in progress

CL: Clearance.

Figure 4. Monocultures or co-cultures (initial numbers shown in Table 3) were seeded into the reactors

After allowing the cultures to stabilize for 3 days, dexamethasone was removed from the medium and 24 h later, 50% the wells were stimulated with 1 µg/ml LPS. Measurements of cytokines and total nitrates/nitrites were made from the medium 48 h after treatment. The results show an enhanced inflammatory response in co-cultures. Dash, Griffith and Tannenbaum, publication in progress. LPS: Lipopolysachcharide.

Figure 5. Monocultures containing a mostly hepatocytes (95%) or co-cultures containing NPCs in defined proportions were seeded into the reactors

After allowing the cultures to stabilize for 3 days, dexamethasone was removed from the medium and 24 h later, 50% the wells were stimulated with 1 µg/ml LPS and/or the test drugs (ranitidine (800 mg/ml), perhexiline (5 mmol), famotidine (175 mg/ml) or DMSO controls). Measurements of LDH release were made from the medium 48 h after treatment. LPS markedly increased toxicity of the known idiosyncratic drug ranitidine but not its corresponding negative control famotidine. Dash, Griffith and Tannenbaum, publication in progress. LDH: Dehydrogenase: LPS: Lipopolysachcharide: NPC: Non-parenchymal cell.